Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Degradation of cartilage proteoglycans was investigated under neutral conditions (pH 7.5) by using pig kidney calpain II (EC 3.4.22.17; Ca(2+)-dependent cysteine proteinase). Aggregate and monomer degradation reached a maximum in 5 min at 30 degrees C when the substrate/enzyme ratio was less than 1000:1. The mode of degradation was limited proteolysis of the core protein; the size of the products was larger than that of papain-digested products and comparable with that of trypsin-digested products. The hyaluronic acid-binding region was lost from the major glycosaminoglycan-bearing region after incubation with calpain II. Calpains thus may affect the form of proteoglycans in connective tissue. Ca(2+)-dependent proteoglycan degradation was unique in that proteoglycans adsorb large amounts of Ca2+ ions rapidly before activation of calpain II: 1 mg of pig cartilage proteoglycan monomer adsorbed 1.3-1.6 mu equiv. of Ca2+ ions before activation of calpain II, which corresponds to half the sum of anion groups in glycosaminoglycan side chains. This adsorption of Ca2+ was lost after solvolysis of proteoglycan monomer with methanol/50 mM-HCl, which was used to desulphate glycosaminoglycans. Therefore cartilage proteoglycans are not merely the substrates of proteolysis, but they may regulate the activation of Ca(2+)-dependent enzymes including calpains through tight chelation of Ca2+ ions between glycosaminoglycan side chains.
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PMID:Characterization of proteoglycan degradation by calpain. 149 24

State analysis of low-sulfated chondroitin 4-sulfate (LSC) in human urine and serum was performed by the use of high performance liquid chromatography and Western blot analysis. It was revealed that the most amount of LSC in urine is present as urinary trypsin inhibitor and a small amount (about 10% of total LSC) is as an LSC chain. The LSC in serum is mainly present as a proteoglycan such as inter-alpha-trypsin inhibitor (ITI), with a molecular weight of 212 kDa, but a small amount of LSC-proteoglycans having molecular weights of 128 and 38 kDa were also observed on SDS-PAGE. Those two compounds may be fragments of ITI, or one of the compounds (128 kDa) may be pre-alpha-trypsin inhibitor which was found by Enghild et al. (J. Biol. Chem., 264, 15975 (1989)).
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PMID:Study on proteoglycans having low-sulfated chondroitin 4-sulfate in human urine and serum. 152 99

Rat ovarian granulosa cells synthesize two distinct species of plasma membrane-intercalated heparan sulfate (HS) proteoglycans; glycosylphosphatidylinositol (GPI)-anchored and core protein-intercalated HS proteoglycans. Both species of HS proteoglycans are primarily localized on the plasma membrane. Cell surface localization of GPI-anchored and protein-intercalated HS proteoglycans can be determined by their accessibility to exogenously added phosphatidylinositol-specific phospholipase C (PI-PLC) and trypsin, respectively. Kinetic parameters for the processes involving their transfer from the Golgi to the cell surface, endocytosis and secretion, and the modes of intracellular degradation were determined by metabolic labeling experiments using [35S]sulfate and various chase protocols in combination with the use of PI-PLC and trypsin in rat ovarian granulosa cells. The experiments demonstrated that (i) both HS proteoglycan species are transferred from the Golgi to the cell surface with an average transit time of approximately 12 min. (ii) GPI-anchored HS proteoglycans are endocytosed with a t1/2 approximately 3 h, without being shed into the medium, and they are rapidly degraded, t1/2 approximately 25 min, without generating recognizable degradation intermediates. (iii) Protein-intercalated HS proteoglycans are partly (approximately 30%) shed from the cell surface into the medium and the remaining approximately 70% are endocytosed with a t1/2 approximately 4 h. After endocytosis, they undergo a slow (t1/2 approximately 4 h) stepwise degradation generating distinct HS oligosaccharides as degradation intermediates. These results indicate that the GPI-anchored and the protein-intercalated HS proteoglycans have distinct secretory, endocytotic, and intracellular degradation pathways probably due to the differences in their anchor structures.
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PMID:Glycosylphosphatidylinositol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotic, and intracellular degradative pathways. 153 36

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.
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PMID:Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern. 154 Dec 70

A very high molecular weight mucin-like glycoprotein was isolated by gel filtration of interphotoreceptor matrix (IPM) from fresh bovine eyes and purified to apparent homogeneity by cesium chloride/guanidine hydrochloride (GuHCl) equilibrium density gradient centrifugation. Although a molecular weight in excess of 10(7) Da is suggested by gel filtration, the presence of SDS or GuHCl did not alter its elution position, indicating that the large size was not simply due to aggregation. Treatment of this material with disulfide reagents, however, led to a decrease in molecular size. On a relative basis, substantially more of this glycoprotein is present in IPM prepared from retina than from retinal pigment epithelium. While the carbohydrate and amino acid composition are not those of a true 'mucin', the large size and many other properties are quite 'mucin-like'. The carbohydrate composition suggests the presence of both N- and O-glycosidically linked sugar chains. The presence of a mucin-type O-glycosidic linkage is indicated by its susceptibility to alkaline cleavage, with concomitant loss of serine and threonine and increase in 240 nm absorbance; production of a fluorescent product upon reaction with cyanoacetamide; lectin binding properties; and production of N-acetylgalactosaminitol upon alkaline borohydride elimination. This glycoprotein was digested by pronase and trypsin, confirming its protein nature, but was resistant to digestion with chondroitin ABC lyase, hyaluronidase and heparinase, as well as RNAase, indicating that these components were not present to any appreciable extent. ELISA for cartilage keratan sulfate was also negative. Centrifugation in CsCl/GuHCl gradients indicated a density much lower than that of a proteoglycan or nucleic acid as well. In vitro biosynthetic studies suggest that both retina and retinal pigment epithelium may be major sources of material in the IPM. The elution patterns of radioactivity were strikingly similar to the UV elution patterns of IPM. The medium from retinal incubations contained very high molecular weight material which was resistant to enzymes which hydrolyse glycosaminoglycans, suggesting that retina may be the source of this high molecular weight, mucin-like glycoprotein.
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PMID:High molecular weight mucin-like glycoproteins of the bovine interphotoreceptor matrix. 154 29

Newly synthesized 35S-labeled chondrocytic keratan sulfate chains were generated by chondrocytes of human chondrosarcoma cell line 105KC and were analyzed for heterogeneity of regional substitution, hydrodynamic size, and charge density. After isolation of the high density large chondrocytic proteoglycans and sequential digestions with chondroitinase ABC, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, and alpha-chymotrypsin, followed by Superose 6 chromatography, two populations of keratan sulfate-containing proteoglycan fragments were identified and pooled separately. Keratan sulfate chains from each of the regions were compared after release by Pronase digestion, and differences in substitution patterns were observed; keratan sulfate chains of greater polydispersity, as well as a population of larger hydrodynamic size, were present in only one of the two regions. Alkaline/borohydride treatment confirmed both the existence of a population of uniquely large keratan sulfate chains and its restriction to a single region of proteoglycan fragments. In addition to heterogeneity of hydrodynamic size, the keratan sulfate chains exhibited regional heterogeneity of charge density and hence, of sulfation patterns. Analysis by Mono Q chromatography identified distinct groups of keratan sulfate that segregated by charge density and whose proportionate composition differed between the proteoglycan regions. Furthermore, the most highly charged species were unique to a single region and encompassed the chains of larger hydrodynamic size. This suggests that there may be regional heterogeneity of keratan sulfate chains substituted along a single class of proteoglycans and identifies a novel population of large, highly sulfated chondrocytic keratan sulfate chains.
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PMID:Heterogeneity of keratan sulfate substituted on human chondrocytic large proteoglycans. 155 68

We characterized the release and the protease composition of high m.w. complexes released from dispersed human skin mast cells, under conditions that did not disrupt the binding of proteases to proteoglycan. The net percent release ratio of tryptase to histamine, after anti-IgE and calcium ionophore A23187 stimulation was higher than those for chymase or carboxypeptidase. This was explained by the greater cell association of carboxypeptidase and chymase, compared with tryptase, after mast cell degranulation and/or differential cosedimentation of the proteases with mast cells, because treatment of activated mast cells with 1 M NaCl increased the release ratios of chymase and carboxypeptidase more than that of tryptase. Tryptase, after release, was stable in 0.12 M NaCl and had a molecular mass of approximately 200 to 250 kDa, suggesting that it was bound to proteoglycan. We demonstrated that complexes containing chymase and carboxypeptidase were separable from tryptase-containing complexes by gel filtration and by affinity chromatography. First, on fast protein liquid chromatography, released tryptase filtered at a molecular mass of approximately 200 to 250 kDa, compared with chymase and carboxypeptidase at 400 to 560 kDa. Second, by using affinity chromatography with immobilized antitryptase mAb in 0.15 M NaCl, carboxypeptidase and chymase activities were recovered primarily in the effluent and washes of an antitryptase antibody affinity column and cofiltered at 400 to 560 kDa. Tryptase was recovered only in the eluate. Finally, by using potato tuber carboxypeptidase inhibitor-Sepharose affinity chromatography, tryptase activity was found primarily in the effluent and washes, filtered at a molecular mass of 200 kDa on fast protein liquid chromatography, and was stable in 0.12 M NaCl buffer at 37 degrees C. Carboxypeptidase and chymase activities were found primarily in the eluate. These findings suggest that tryptase and carboxypeptidase/chymase reside in distinct macromolecular complexes. Separate complexes containing these proteases may help explain previous ultrastructural observations in which the distributions of chymase and tryptase within a single granule did not always coincide.
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PMID:Protease composition of exocytosed human skin mast cell protease-proteoglycan complexes. Tryptase resides in a complex distinct from chymase and carboxypeptidase. 156 Feb 3

The binding of a 34-kDa (mol. wt.) acylpoly(1,3)galactoside (APG) extracted from a membrane proteoglycan of Klebsiella pneumoniae to human blood leucocytes was investigated. APG is made of a long poly(1,3)galactose chain, a core-like region and a lipid moiety which comprises two glucosamine residues bound to a phosphate group and two beta OH myristic acids. Fluoresceinated APG was shown to bind preferentially to monocytes and to a lesser extent to polymorphonuclear neutrophils, as determined by flow cytometry. Binding of fluoresceinated APG was inhibited by unlabelled APG; it was concentration dependent, but not saturable, with rapid kinetics. It occurred at +4 degrees C but was markedly increased at 37 degrees C. It involved trypsin-sensitive molecules on the membrane of monocytes. Neither the parent proteoglycan nor lipopolysaccharide from K. pneumoniae or Salmonella minnesota competed for APG binding. A minor non-specific binding to lymphocytes, occurring predominantly on B cells, was observed. Unlike that of lipopolysaccharide, the APG binding was not blocked by polymyxin B sulphate. Interaction between the galactose chain of APG and the galactose receptor does not account for the binding of APG to monocytes because the galactose receptor (Mac-2) is expressed at high density on activated macrophages but not on monocytes. Despite its strong binding to human blood monocytes, APG displayed a much weaker activity than K. pneumoniae membrane proteoglycan with respect to induction of monocyte cytokine synthesis. When administered as a Technetium 99 conjugate, APG was shown to label inflammatory foci in experimental animals, and its property as a marker of macrophages is currently being evaluated in clinical trials.
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PMID:Binding of a bacterial acylpoly(1,3)galactoside to human blood leucocytes. 161 80

1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had an apparent molecular mass of 18 kDa. Results of heparitinase digestion and HNO2-treatment indicated a clustering of sulfate groups in the distal part of the HS side chains. 5. Peptide mapping after trypsin, clostripain or V8 protease digestion of radiolabeled human and equine heparan sulfate proteoglycans (HSPG) preparations with three different separation techniques showed large differences. 6. Polyclonal antisera raised against the HSPGs reacted against the core proteins. Both HSPG preparations and their antisera showed ca 40% cross-reactivity. About 50% of monoclonal antisera elicited against one HSPG preparation showed reaction with both HSPG preparations. 7. Polyclonal antisera stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies of kidney sections from horse, man and various mammalian species. 8. Biochemical and immunological data indicate that HSPGs from equine and human GBM have a comparable structure, but the core proteins differ considerably.
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PMID:Comparison of heparan sulfate proteoglycans from equine and human glomerular basement membranes. 170 71

Cultured rat glomerular epithelial cells (GEC) are able to prevent both antibody-directed and spontaneous (alternative pathway) complement activation. In this study, a novel complement regulatory factor (GCRF) was isolated from GEC. The ability to accelerate the decay of alternative pathway C3/C5 convertases formed on sheep erythrocytes (EC3bBbP) was used to guide purification. GEC were solubilized in Triton X-114 and GCRF was recovered in the aqueous phase. Complement inhibitory material also was present in the culture supernatant, which likely represented GCRF. By Mono Q anion exchange chromatography, GCRF eluted at greater than or equal to 0.6 M NaCl and by Superose 6 size-exclusion chromatography, it had a Kav less than or equal to 0.3. GCRF reduced the t1/2 of EC3bBbP from 128 minutes in buffer alone to 41 minutes in 3 micrograms/ml GCRF protein, and also prevented formation of EC3bBbP in a dose-dependent fashion. Digestion with chondroitinase ABC, neuraminidase, or trypsin, but not with heparitinase or chondroitinase AC significantly reduced the activity and size of GCRF, demonstrating that it is a sialic acid-containing dermatan sulfate proteoglycan. Thus, cultured rat GEC synthesize and secrete into the medium, GCRF, a dermatan sulfate proteoglycan with complement inhibitory activity.
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PMID:Isolation of a novel complement regulatory factor (GCRF) from glomerular epithelial cells. 174 16


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